Haemophilus — “blood-loving”

More than 100 years ago, in 1892, German bacteriologist Richard Pfeiffer isolated from patient nasal secretions a bacillus he thought was the causative agent of influenza, which he called Bacillus influenza. Subsequently, “Pfeiffer’s bacillus” was considered the cause of the 1918 Spanish flu until, in 1933, following outbreaks in pigs in Iowa two years earlier, smaller, filterable particles – i.e. a Myxovirus—were implicated. Bacillus influenza got a name change to Haemophilus influenzae, bearing the mark still of its putative link to the flu.

Before the era of Hib vaccination, H. influenza was notorious for causing the feared infections of epiglottitis and meningitis. The organism is a recognised opportunist, gaining a foothold in those infected with Myxovirus (e.g. influenza), for instance, explaining its frequent isolation during outbreaks of flu.

But H. Influenzae augments the flu and can lead to complications of:

• pneumonia
• meningitis
• otitis media

Slightly larger than S. aureus, the Gram-negative Haemophilus coccobacillus remains a difficult organism to identify as it only stains weakly and is often confused with Neisseria species. It will not routinely grow on blood agar, since the V-factor in red cells is not readily available. Instead, Chocolate agar, a form of blood gar where the V-factor has been released, and when components in blood that could inactivate it are destroyed, is employed to culture the fastidious organism.

Haemophilus Growth Remains Dependent Upon Two Essential Nutrients

1. Heat-stable X-Factor (haemin or haematin)
2. V-Factor (nicotinamide adenine dinucleotide or NAD), a cofactor central to metabolism found in all living cells and commonly made available in laboratory samples through yeast extract

Because of this, H. influenza is said to show the characteristic of satellitism — it will grow on blood agar only nearby colonies of Staph. aureus. The blood (agar) provides the X-factor, and co-located colonies of S. aureus provide the V-factor.

Virulent strains of H. influenza are characterised according to their capsular antigen.

The polyribophosphate (PRP) capsule of H. influenza is poorly immunogenic in children under two years of age. It is this age group that the life-threatening infections of epiglottitis and meningitis were most frequent; older children and adults have antibodies to H. Influenzae. Until recently, attempts to immunise these younger children against Haemophilus had failed. Current vaccines conjugate (link) the PRP to a protein, such as tetanus toxoid, rendering it T-cell dependent and more immunogenic.

The capsular antigen stimulates IgM, production of which is largely T-cell independent, yet lgM does not diffuse into the cerebrospinal fluid (CSF) like the T-cell dependent but smaller IgG molecule, which can penetrate into the CSF.

Who is Susceptible to Infection with Haemophilus?

1. Males > Females: females are better antibody producers
2. Children < 2 years old
3. Sickle-cell anaemia
4. Splenectomy or congenital aplasia of spleen
5. Agammaglobulinaemia
6. Alcoholism

H. influenzae
	Otitis media can potentially spread to the meninges and meningitis can also seed from other sites e.g. pneumonia (i.e. via haematogenous spread into the CSF).
H. parainfluenzae

The carriage rate of virulent strains of H. influenza is about 2% of the population throughout the year but the incidence of serious infection is greatest in winter-spring, following other viral respiratory infections. Incidence of H. influenzae meningitis is inversely proportional to bactericidal antibodies in serum. The organism produces an extracellular enzyme, lgA1 protease, which cleaves and renders ineffective the IgA mucosal antibody. The more prevalent non-capsulated strain of H. influenza forms part of most normal upper respiratory tract flora but is nonetheless strongly associated with episodes of bronchitis, filling the lumen of bronchi with inflammatory exudate.

(Previously, an oral vaccine, ‘Bronchostat’, made of 10⁹ killed H. Influenzae organisms, was used to induce a local immunity (predominantly IgA) that interferes with the ability of H. Influenzae to become established.)

H. Influenzae also cause sinusitis, otitis media, and conjunctivitis. Importantly, with many H. Influenzae infections, bacteraemia is a constant feature and blood cultures are most productive.

Diagnosis

Obtain the following specimens:

• Nasopharyngeal swab and sputum
• Blood

Smear specimens onto microscopic slide, Chocolate (X & V-factors) and Blood Agars:

1. Gram stain
2. Culture – H. influenzae grow as translucent colonies
3. Satellitism – Blood Agar with S. aureus

Treatment

Traditionally, amoxycillin given together with chloramphenicol were used because 5-15% of H. influenza strains are beta-lactamase producers, ceasing the latter once sensitivities were confirmed. In today’s Hib vaccination era, in the unlikely event of Haemophilus epiglottitis or meningitis, a third generation cephalosporin, like ceftriaxone or cefotaxime, at a dose of 50 mg/kg per day, should suffice for treatment.

Serious H. influenzae infections

Epiglottitis

The child presents with:

• Tense and frightened with difficulty breathing
• Swelling at back of tongue
  • X-Rays reveal this (epiglottis larger than thumb)
• Laryngitis
• May go into shock
• May need tracheotomy
  • Can be life-threatening
• Usually H. influenzae type b (Hib)

Meningitis

Again, chloramphenicol was added as back-up in case the organism was resistant to ampicillin, i.e. a beta-lactamase producer. Other broad-spectrum antibiotic, like gentamycin, were also used. Today, a third generation cephalosporin is first-line treatment.

Other Serious Gram Negatives

Moraxella

• Gram-negative coccobacilli
• Waterborne, non-motile
• M. lacunate are a common cause of an angular bacterial conjunctivitis

Acinetobacter

• Gram-negative very small cocci, similar to or smaller in size than S. aureus
• Opportunist pathogen – e.g. Ac. anitratus
  • Post-operative wound infections
  • UTIs
  • Pneumonia
  • Septicaemia
• Not to be confuses with early presumptive diagnosis of gonorrhoea, as N. gonorrhoea is similar

• Very small, waterborne
  • Can get through filtration systems
    • Deaths from contaminated IV fluids
• Quickly precipitates shock (Gram-negative sepsis)

Veillonella

• Gram-negative obligate anaerobic coccus
• Part of normal flora of mouth and gut
• Opportunist: causes serious infection in the right circumstances
  • V. parvula similar to Bacteroides infections

Neisseria

• Gram-negative, reniform (kidney-like) morphology
• Aerobic
• Nutritionally fastidious
  • do not grow on ordinary Nutrient or Blood Agar
  • will grow on Chocolate agar
• Distinguished by differential effect on sugars – differing abilities to ferment maltose
• Similar to Haemophilus – produce IgA1 protease (extracellular enzyme) whose significance remains unknown

Persons who suffer recurrent infections of gonorrhoea have antibodies to IgA1 protease (i.e. not protective), although IgA1 protease is now known to destroy human IgA1 thereby eliminating an element of host defence.

• Have pili – to adhere to cells

Differentiation of Neisseria species based on fermentation of sugars

Species	Glucose	Maltose	Sucrose	Lactose
N. gonorrhoea	+	–	–	–
N. meningitides (capsulated)	+	+	–	–

N. gonorrhoea

• fever
• chills
• polyarthralgia

1. Adhere to epithelial cells – pili
2. Gonococcus endocytosed and vacuolated
3. Exocytosed
4. Intracellular – can  escape effects of immune defences (especially antibodies) and upon their exocytosis they often hide within host antigens, enclosing them – i.e. upon their exocytosis host antigens attach to its surface and not recognised as non-self.

N. meningitides (meningococcus)

• Severe life-threatening
• Rapidly acquired and rapidly progressive
  • By the time one child is treated, another child in the family can get it and within 24 hours both could be dead
• Often serious side effects if survive
  • Mental retardation
  • Defects in hearing, speech, vision
• Uniformly fatal left untreated (as is H. Influenzae infection)
  • Rx: Chloramphenicol + Penicillin / Ampicillin / Gentamycin
  • Even treated, mortality up to 30-40%
  • Family members given prophylactic antibiotics – Rifampicin
• Development of rash
  • Due to effects of antigen provoking inflammatory reaction

1. Can metabolise lactoferrin as a source of iron
2. Gram stain CSF
3. Organism can be isolated from (maculopapular skin) lesions: this is one of the features of meningococcaemia
4. Notifiable Disease
5. Three capsular types responsible for epidemics of meningitis and associated with inter-epidemics, especially in India
6. Organism can be β-lactamase producer – otherwise, previously, withdrew chloramphenicol from combined treatment with ampicillin: today, use third generation cephalosporin
7. Vaccine least effective in those most susceptible – i.e. children < 2, in whom the polysaccharide remains poorly immunogenic

Immunisation against Meningococcal Disease

The first vaccine against meningococcus was introduced 50 years ago. Antibody response to these purified capsular polysaccharide vaccines declines rapidly over months in young children and “the global burden of invasive meningococcal disease remains substantial, lingering, and unpredictable”._¹ Conjugating the polysaccharide to diphtheria or tetanus toxoid affords a more durable immune response:

• MenACWY-TT vaccine can be administered from 6 weeks of age
• MenACWY-D can be administered from 9 months of age
• MenB-4C can be administered from 2 months of age
• MenB-fHbp can be administered from 10 years of age

Conjugation of capsular polysaccharide has afforded younger and wider coverage for immunisation against the dual childhood scourges of Haemophilus influenza (Hib) and Neisseria meningitides infection.

References

Knuf, Markus. Performance of Modern Meningococcal Vaccines: The Accumulated Evidence & Clinical Implications. Medscape. Available at http://www.medscape.org/viewarticle/935756.

Mistry, Dippica and Stockley, Robert A. “Molecules in Focus: IgA1 protease.” The International Journal of Biochemistry & Cell Biology 38(8), 2006: 1244-1248. https://doi.org/10.1016/j.biocel.2005.10.005.

Todar, Kenneth. “Nutrition and Growth of Bacteria (page 2).” Todar’s Online Textbook of Bacteriology. 2020. Available at http://textbookofbacteriology.net/nutgro_2.html. Accessed September 8, 2020.

Van Epps, Heather L. “Influenza: exposing the true killer.” J Exp Med. 2006;203(4): 803. doi: http://10.1084/jem.2034fta.